A novel two-photon-fluorescent N,O-heteroatom-rich carbon nanomaterial has been synthesized and characterized. The new carbon nanoparticles were produced by hydro-thermal conversion from a one-photon-fluorescent poly-(4-vinylpyridine) precursor (P4VP). The carbonized particles (cP4VP-dots) with non-uniform particle diameter (ranging from sub-6 nm to 20 nm with some aggregates up to 200 nm) exhibit strong fluorescent properties in different solvents and have also been investigated for applications in cell culture media. The cP4VP-dots retain their intrinsic fluorescence in a cellular environment and exhibit an average excited state lifetime of 2.0 ± 0.9 ns in cell. The cP4VP-dots enter HeLa cells and do not cause significant damage to outer cell membranes. They provide one-photon or two-photon fluorescent synthetic scaffolds for imaging applications and/or drug delivery.